Dermal filler

ABSTRACT

Improved dermal filler formulation comprising a hyaluronic acid and a botulinum toxin.

RELATED APPLICATION

This application is related to U.S. patent application Ser. No.11/828,561 filed Jul. 27, 2007.

BACKGROUND

The present invention relates to an improved dermal filler. Inparticular the present invention relates to an injectable, highviscosity dermal filler formulated with a botulinum toxin.

A pharmaceutical composition (synonymously a formulation or acomposition) is a formulation which contains at least one activeingredient (for example a botulinum neurotoxin) as well as, for example,one or more excipients, buffers, carriers, stabilizers, preservativesand/or bulking agents, and is suitable for administration to a humanpatient to achieve a desired effect or result. The pharmaceuticalcompositions disclosed herein have cosmetic utility.

Movement of the face due to contractions of muscles underlying the skincan result in facial wrinkles. For example, repeated elevation of thebrow by contraction of the frontalis muscle can cause brow furrows.Dermal fillers made from various substances have been used to treatfacial wrinkles. Collagen based dermal fillers include ZYDERM®(Allergan, Inc., Irvine, Calif.), ZYPLAST® (Allergan, Inc., Irvine,Calif.), COSMODERM® (Allergan, Inc., Irvine, Calif.), COSMOPLAST®(Allergan, Inc. Irvine, Calif.) and Autologen (Collagenesis, Inc., BocaRaton, Fla.). Polylactic acid dermal fillers include SCULPTRA®(Sanofi-Aventis, Bridgewater, N.J.). Calcium hydroxyl apatite dermalfillers include RADIESSE® (BioForm Medical, Inc., San Mateo, Calif.).Hyaluronic acid dermal fillers include HYLAFORM® (Genzvme Corp.,Cambridge, Mass.), RESTYLANE® (HA North America Sales AB, Scottsdale,Ariz.) and JUVEDERM® (Allergan, Inc., Irvine, Calif.). JUVEDERM®comprises a sterile, biodegradable, non-pyrogenic, viscoelastic, clear,colorless, homogenized gel consisting of cross-linked hyaluronic acidformulated at a concentration of 24 mg/ml in a physiologic buffer.

Electromyographic methods have been used to study the activity ofvarious facial muscles which can cause facial wrinkles. See e.g.Fridlund A. et al., Guidelines for Human Electromyographic Research,Psychophysiology 1986; 23(5): 567-590; Vitti M, et al.,Electromyographic Investigation of Procerus and Frontalis Muscles,Electromyogr. Clin. Neurophysiol. 1976, 16: 227-236, and; Tassinary L.et al., A Psychometric Study of Surface Electrode Placements for FacialElectromyographic Recording: I. The Brow and Cheek Muscle Regions,Psychophysiology 1989; 26(1): 1-16.

In particular, electromyography, including surface electromyography(sEMG) has been used to investigate activity of the frontalis muscle andresultant brow displacement. See e.g. van Boxtel A, et al., Amplitudeand bandwidth of the frontalis surface EMG: Effects of electrodeparameters, Psychophysiology 1984; 21(6): 699-707, and; Pennock J. D.,et al., Relationship between muscle activity of the frontalis and theassociated brow displacement, Plast Reconstr Surg November 1999; 104(6):1789-1797.

Additionally, it is known to study skin topography (i.e. facialwrinkles) by making a silicone rubber negative replica (a mold) of askin surface area. The mold captures three dimensional details of theskin surface and computerized image analysis of skin line density,depths and length analysis shown can be carried out thereon. Grove, G.L., et al, Objective method for assessing skin surface topographynoninvasively, chapter one, pages 1-32 of Cutaneous Investigation inHealth and Disease, edited by Leveque J-L., Marcel Dekker, Inc. (1989).The same silicone rubber impression method has been used to examine theeffect of a topical cream to treat photodamaged skin, as by reduction ofperiorbital (crows feet) wrinkles. Leyden J. J., et al., Treatment ofphotodamaged facial skin with topical tretinoin, J Am Acad Dermatol1989; 21(3) (part 2): 638-644, and; Grove G. L., et al., Skin replicaanalysis of photodamaged skin after therapy with tretinoin emollientcream, J Am Acad Dermatol 1991; 25(2) (part 1): 231-237. See also U.S.Pat. Nos. 6,688,311; 7,234,469, and; 7,140,371.

Hyaluronic Acid

Hyaluronic acid (also called hyaluronan or sodium hyaluronate) is anaturally occurring polysaccharide found in joints, connective tissueand the eye. Hyaluronic acid is a glycosaminoglycan (amucopolysaccharide) which is a long unbranched polysaccharide composedof repeating dimeric units of glucuronic acid and N acetyl glucosamine.U.S. Pat. Nos. 4,636,524; 4,713,448; 5,099,013, and 5,143,724 discloseparticular hyaluronic acids and methods for making them.

Hyaluronic acid has known therapeutic and cosmetic uses. For example,intra-articular use of hyaluronic acid as a viscosupplement to treatosteoarthritis joint pain is known (eg ORTHOVISC® (Anika, Woburn,Mass.), DUROLANE® (Q-Med AB, Uppsala, SE/Smith & Nephew, Rotkreuz, CH),HYALGAN® (Sanofi-Aventis, Bridgewater, N.J.), Hylastan (Genzyme Corp.,Cambridge, Mass.), SUPARTZ® (Seikagaku, Tokyo, JP/Smith & Nephew,Rotkreuz, CH), SYNVISC® (Genzyme Corp., Cambridge, Mass.), and EUFLEXXA®(Ferring B.V., Hoofddorp, NL)). Hyaluronic acid is also usedcosmetically as an injectable soft tissue dermal filler (eg JUVEDERM® )to treat facial rhytides.

The duration of the wrinkle alleviating effect of action of a dermalfiller is typically about one year or less, and can be only a few monthsfor eg dermal fillers administered in a perioral area. The duration ofthe wrinkle alleviating effect of action of a hyaluronic acid dermalfiller is influenced by mechanical stress upon enzymatic degradation ofthe hyaluronic acid, as well as by the rate of clearance of thehyaluronic acid from the site of injection via the extensive tissuelymphatic vessels. Mechanical stress degradation of the injectedhyaluronic acid is especially notable in areas of the face whichexperience frequent muscle contraction, such as around the mouth, eyesand the forehead. In particular, the dermis overlying the orbicularisoris muscle in the perioral area is a site of high mechanical stress dueto frequent muscle contraction. Such repetitive mechanical stress canlead to movement of the injected hyaluronic acid from the intended siteof action and untimely (premature) degradation of the dermal fillerpolymer.

U.S. patent applications which disclose use of therapeutic agentformulated with a hyaluronic acid include application Ser. No.10/966,764, filed Oct. 14, 2004, application Ser. No. 11/091,977, filedMar. 28, 2005, application Ser. No. 11/354,415, Feb. 14, 2006,application Ser. No. 11/741,366, filed Apr. 27, 2007, application Ser.No. 11/828,561, filed Jul. 26, 2007, application Ser. No. 11/039,192,filed Jan. 19, 2005, application Ser. No. 11/116,698, filed Apr. 27,2005, application Ser. No. 11/695,527, filed Apr. 2, 2007, andapplication Ser. No. 11/742,350, filed Apr. 30, 2007.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin called botulinum neurotoxin toxin whichcauses a neuroparalytic illness in humans and animals referred to asbotulism. Botulinum toxin type A is the most lethal natural biologicalagent known to man. About 50 picograms of a commercially availablebotulinum toxin type A (purified neurotoxin complex)¹ is a LD₅₀ in mice(i.e. 1 unit). One unit of BOTOX® contains about 50 picograms (about 56attomoles) of botulinum toxin type A complex. One unit (U) of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing 18 to 20 grams each. Available fromAllergan, Inc., of Irvine, Calif. under the tradename BOTOX® in 100 unitvials)

Seven, generally immunologically distinct botulinum neurotoxins havebeen characterized, these being respectively botulinum neurotoxinserotypes A, B, C₁, D, E, F and G each of which is distinguished byneutralization with type-specific antibodies. The different serotypes ofbotulinum toxin vary in the animal species that they affect and in theseverity and duration of the paralysis they evoke. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

Botulinum toxins have been used for the treatment of various therapeuticand cosmetic conditions. A botulinum toxin type A (Allergan, Inc.,BOTOX®) has been approved by the U.S. Food and Drug Administration forthe treatment of blepharospasm, strabismus, cervical dystonia,hyperhydrosis and glabellar lines.

The molecular weight of the neurotoxic component of a botulinum toxincomplex is about 150 kD. Botulinum toxin is typically made by theClostridial botulinum bacterium as a complex comprising the 150 kDbotulinum toxin protein molecule and associated non-toxin proteins.Thus, a botulinum toxin type A complex can be produced by Clostridialbacterium as 900 kD, 500 kD and 300 kD complex forms.

It is known that injection of a botulinum toxin into facial muscles can,by weakening the injected muscles, result in a decrease of hyperkineticwrinkles in the skin overlying the paralyzed muscles. See e.g.Carruthers A. et al., The treatment of glabellar furrows with botulinumA exotoxin, J Dermatol Surg Oncol January 1990;16(1):83. Thus, botulinumtoxin has been injected into facial muscles, such as the orbicularisoculis, corrugator supercilii and frontalis muscles for the cosmeticpurpose of reducing certain facial wrinkles, and it is known to useelectromyographic and/or photographic techniques to assess the efficacyof such injections. Guerrissi J. et al., Local injection into mimeticmuscles of botulinum toxin A for the treatment of facial lines, AnnPlast Surg 1997;39(5):447-53. Electromyography has also been used toassess the effect of injection of a botulinum toxin into thesternocleidomastoid muscle for treatment of cervical dystonia. DresslerD. et al., Electromyographic quantification of the paralysing effect ofbotulinum toxin in the sternocleidomastoid muscle, Eur Neurol 2000; 43:13-16. In sEMG the surface electrodes are placed at fixed distances fromthe injection point, typically 1 cm and 3 cm from the injection point.The surface electrodes can be used to measure the amplitude and area ofa compound muscle action potential (CMAP) during maximal voluntarycontraction of the injected muscle. One expects to find that CMAPdecreases with the onset of muscle paralytic effect and to increase asthe paralytic effect wears off.

Photographic methods, such as digital image analysis, have been used todetermine efficacy of a botulinum toxin to treat hyperkinetic faciallines. Heckmann M., et al., Quantification of the efficacy of botulinumtoxin type A by digital image analysis, J Am Acad Dermatol 2001; 45:508-514.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative. Othercommercially available botulinum neurotoxins approved for use in humansinclude DYSPORT® (Beaufour Ipsen, Porton Down, England) XEOMIN® (MerzPharmaceuticals GmbH, Frankfurt, Germany) and MYOBLOC® (SolsticeNeurosciences, San Francisco, Calif.).

What is therefore needed is an improved dermal filler formulation whichhas a longer duration (i.e. more than three months in a perioral area)wrinkle alleviating effect upon injection (subdermal) of the dermalfiller, for example by decreasing the mechanical stress and enzymaticdegradation of the injected dermal filler.

SUMMARY

The present invention meets this need and provides a dermal fillerformulation which has a longer duration wrinkle alleviating effect uponinjection of the dermal filler.

Definitions

As used herein, the words or terms set forth below have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration” or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject. Thepharmaceutical compositions disclosed herein are “locally administered”,that is administered at or in the vicinity of the site at which atherapeutic result or outcome is desired. For example to treat anperipheral condition by peripheral administration of a viscousformulation. “Sustained release” means release of an active agent (suchas a botulinum neurotoxin) over a period of about seven days or more,while “extended release” means release of an active agent over a periodof time of less than about seven days.

“Botulinum toxin” means a botulinum neurotoxin type A, B, C, D, E, F orG as either pure toxin (i.e. the about 150 kiloDalton molecular weightneurotoxic component) or as a botulinum toxin complex (about 300 toabout 900 kiloDaltons molecular weight), including recombinant,chimeric, hybrid, retargeted, and amino acid sequence modified botulinumneurotoxins, but excluding botulinum toxins which are not neurotoxinssuch as the cytotoxic botulinum toxins C₂ and C₃.

“Entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed, or that thecondition, complication or side effect does not exist.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected, or that the condition,complication or side effect is experienced by a patient for less than10% to 20% of the time.

“Facial wrinkle” includes both a wrinkle or rytide on the head (i.e.face, cheeks, etc) or neck as well as dermal furrows, depressions, pits,craters and sunken skin anywhere on the head or neck of a patient.

“Local administration” means administration (i.e. by a subcutaneous,intramuscular, subdermal or transdermal route) of a pharmaceutical agentto or to the vicinity muscle or a subdermal location by a non-systemicroute. Thus, local administration excludes systemic (i.e. to the bloodcirculation system) routes of administration, such as intravenous ororal administration. Peripheral administration means administration tothe periphery (i.e. to a location on or within a face, limb, trunk orhead of a patient) as opposed to a visceral or gut (i.e. to the viscera)administration.

“Pharmaceutical composition” means a formulation in which an activeingredient (the active agent) can be a botulinum neurotoxin. The word“formulation” means that there is at least one additional ingredient inthe pharmaceutical composition besides the active agent. Apharmaceutical composition is therefore a formulation which is suitablefor diagnostic or therapeutic administration (i.e. by subdermal orintramuscular injection) to a subject, such as a human patient.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition, or that the condition,complication or side effect is experienced by a patient less than 50% ofthe time.

“Sustained release” means release of an active agent (such as atriamcinolone) over a period of about seven days or more, while“extended release” means release of an active agent over a period oftime of less than about seven days.

“Viscous carrier” means a biocompatible compound which when formulatedwith a botulinum neurotoxin provides upon in vivo local, subdermalinjection of the formulation a depot from which the botulinum toxin isreleased from or diffuses away from at a rate slower than the rate ofrelease or diffusion of the botulinum toxin from a purely aqueoussolution of the botulinum toxin.

All the viscosity values set forth herein were determined at 25° C.(unless another temperature is specified). Additionally, all theviscosity values set forth herein were determined at a shear rate ofabout 0.1/second (unless another shear rate is specified).

Our invention encompasses a method for treating a facial wrinkle byadministering to the patient a pharmaceutical composition comprising abotulinum neurotoxin and a viscous carrier for the botulinum neurotoxin,wherein the patient's facial wrinkles are alleviated for a longer periodof time than they are by administration of a pharmaceutical compositionwhich does not comprise a botulinum toxin. The administering can becarried out by local subdermal injection of the pharmaceuticalcomposition. The disease or condition treated can be a dystonia (such asa cervical dystonia), blepharospasm, strabismus, spasticity, movementdisorder, headache, migraine, hyperhydrosis, overactive bladder,prostate disorder, articular pathology, arthritis, facial wrinkles, andglabellar lines.

The viscous carrier can be selected from the group of viscous carriersconsisting of hyaluronic acid, carbomer, polyacrylic acid, cellulosepolycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharide,polyacrylamide, polyvinyl alcohol, polyvinyl acetate, chitosans,algenates and derivatives and mixtures thereof. Preferably, the viscouscarrier is a hyaluronic acid, such as a non-cross linked hyaluronic acidor a cross linked hyaluronic acid, or a mixture thereof. The hyaluronicacid used can be a polymeric hyaluronic acid with a molecular weightbetween about 10,000 Daltons and about 20 million Daltons and theconcentration of hyaluronic acid in the formulation can be between about0.1 wt % and about 99 wt %.

In our method the period of alleviation of the facial wrinkles can bedetermined by a method chosen for the group of method consisting offacial mould, electromyographic (EMG) recording and photography.

Significantly, the period of time of alleviation of the facial wrinklesis from about 20% to about 100% longer than the period of time thepatient's facial wrinkles are alleviated by administration of apharmaceutical composition injected in the same volume into the samepatient at the same location, to treat the same facial wrinkles, andwhich contains the same amount of the botulinum toxin, but which doesnot comprise a viscous carrier.

The viscosity of the pharmaceutical composition used to practise themethod set forth above can be between about 100 cps and about 300,000cps at 25° C., at a shear rate of 0.1/second. The botulinum neurotoxinused in our improved dermal filler can be a botulinum neurotoxin type A.

A detailed embodiment of a method within the scope of our invention is amethod for treating a facial wrinkle by local, subdermal administeringto the patient a pharmaceutical composition comprising a botulinumneurotoxin type A and a polymeric, hyaluronic acid for the botulinumneurotoxin, wherein the patient's facial wrinkles are alleviated for aperiod of time which is from about 20% to about 100% longer than theperiod of time the patient's facial wrinkles are alleviated byadministration of a pharmaceutical composition injected in the samevolume into the same patient at the same location, to treat the samefacial wrinkles, and which contains the same amount of the botulinumtoxin type A, but which does not comprise the hyaluronic acid.

DRAWINGS

FIG. 1 is a two dimensional magnetic resonance image taken 6 hours afterinjection in an anesthetized rat with the cross-linked hyaluronic acid(Juvederm™) albumin-gadolinium contrast agent complex of Example 7. TheFIG. 1 image is oriented longitudinal through the gastrocnemius muscleto show the tripennate gastrocnemius lateralis (MGL) muscle group wherethe injection with hyaluronic acid-contrast agent complex was performed(arrow in FIG. 1). FIG. 1 shows that there was no movement of thecomplex during the 6 hour anesthesia period.

FIG. 2 is a two dimensional magnetic resonance image taken 28 hoursafter injection in an anesthetized rat. The FIG. 2 image is in the sameorientation as FIG. 1. After the animal was awake and ambulating (i.e.causing muscle contraction in the gastrocnemius), there was migration ofthe hyaluronic acid-contrast agent complex from the initial site ofinjection (small arrow in FIG. 2) towards the Achilles tendon (largearrow in FIG. 2).

DESCRIPTION

The present invention is based on the discovery of a dermalfiller-botulinum toxin formulation which can significantly increase theduration of action of a soft tissue dermal filler in areas of highmechanical stress, that is in areas of facial wrinkles.

We determined that simultaneous injection of a viscous carrier (such asa hyaluronic acid) and a botulinum toxin (such as a botulinum neurotoxintoxin type A, B, C,D, E, F or G) in a combined formulation can causeessentially immediate muscle paralysis and thereby limit mechanicalstress to a polymeric viscous carrier. Other neurotoxins such as atetrodotoxin can also be used in our improved dermal filler formulation.

The improved dermal filler formulation we developed comprises abotulinum toxin and a high viscosity, polymeric carrier for thebotulinum toxin. The viscous carrier can have the characteristic of highshear thinning so that it is injectable through a 25 to 32 gauge needle.

The viscous carrier in our formulation can be a high molecular weight,polymeric, hyaluronic acid. Hyaluronic acid is a major component of theextracellular matrices of soft tissues, such as the skin and muscle. Lowmolecular weight (about 1000 Daltons or less) hyaluronic acid can becleared rapidly from an intradermal or intramuscular location via thelymphatics. A higher molecular weight hyaluronic acid can have a longertissue residence time and be eliminated from a subdermal site throughlower local enzymatic process. We determined that the tissue residencytime of a hyaluronic acid in soft tissues can be increased by reducingboth lymphatic clearance and that this can be accomplished by increasingthe biological stability of the polymer, such as by using particularmethods and cross linkers to cross link a low molecular weighthyaluronic acid to thereby prepare a much higher molecular weight (1million Daltons or higher) hyaluronic acid, which has increasedbiological stability (meaning that the hyaluronic acid takes longer tobiodegrade), is biocompatible and functions as a depot which contains toprevent diffusion of the botulinum neurotoxin formulated with thehyaluronic acid in any amounts which exceed the amount of botulinumtoxin required to saturate adjacent neuromuscular junctions.

We determined that increasing the molecular weight of the polymericviscous carrier concomitantly increases the local botulinum neurotoxinconcentration at or near the site of injection. Additionally, wedetermined that by increasing the degree of cross linking of thehyaluronic acid, or increasing the concentration of a hyaluronic acid inthe formulation or increasing the molecular weight of the hyaluronicacid used in the formulation results in a longer tissue residence timeof the formulation and reduce diffusion of the botulinum neurotoxin fromthe formulation to adjacent muscle groups and importantly also reducessystemic complications form the botulinum neurotoxin, as compared toinjections of aqueous formulations of the same concentration of the samebotulinum neurotoxin at the same subdermal injection site.

In our formulation a preferred viscous carrier is a hyaluronic acid witha molecular weight of between about 10,000 Daltons and about 20 millionDaltons. A preferred total hyaluronic acid concentration of bothcrosslinked and uncrosslinked components in our formulation is fromabout 1 wt % to about 5 wt % cross linked hyaluronic acid. Where a crosslinked hyaluronic acid is used the actual crosslinking density (i.e. howmany of the hyaluronic acid monomers are actually linked to otherhyaluronic acid monomers) is from about 5% to about 40%.

A neurotoxin, such as a botulinum neurotoxin A and/or a tetrodotoxin,can be encapsulated in the polysaccharide polymer network itself or intolarge vessels within the network that enable local, sustained andcontrolled release of actives to the surrounding tissue of the injectionsite. Parameters such as hyaluronic acid concentration, degree ofcrosslinking, median particle size, and molecular weight of the rawmaterial hyaluronic acid can be varied to permit altered releasekinetics for the neurotoxin. The encapsulation can be carried out bysimple mixing or by allowing the neurotoxin to mix with the polymernetwork at a highly hydrated state; followed by dehydration of thenetwork to control the release kinetics (e.g. final swelling ratio ofthe polymer). The contracted network can be sized into particles, mixedwith the dermal filler and delivered at the site of the injection. Theslow re-hydration of the neurotoxin loaded polysaccharide particles canprovide a sustained and controlled delivery of the neurotoxin.

A second method of encapsulating the neurotoxin is to incorporate theactive molecules into biocompatible and biodegradable vessels that couldbe delivered at the same time with the polymer filler. Such vessels canbe composed of non-covalently or covalently linked self-assembledmolecules (e.g. liposomes, polymerized vesicles).

Our improved dermal filler formulation has the following characteristicswhich characteristics provide an improved, injectable dermal filler gelformulation:

-   (1) high rheological strength (G′>300 at 5 Hz at linear viscoelastic    regime);-   (2) when a hyaluronic acid is used as the viscous carrier, the    hyaluronic acid has in the formulation a concentration of between 20    to 40 mg/ml;-   (3) the degree of cross linking of the polymer which comprises the    viscous carrier is between about 1% to about 8% (w/w);-   (4) the weight percent of a cross linker viscous carrier (i.e.    cross-linked hyaluronic acid) in the formulation is at least 85 wt    %;-   (5) the raw material viscous carrier (i.e. a hyaluronic acid) has a    molecular weight of between about 600 kDa and about 1,500 kDa, and;-   (60 the average molecular weight of the total viscous carrier    (soluble non-cross linked viscous carrier plus cross linked viscous    carrier) in the final formulated improved dermal filler is greater    than 400 kDa.

A viscous carrier within the scope of our invention can have a viscosityat 25° C. of at least about 10 cps or at least about 100 cps or at leastabout 1000 cps, more preferably at least about 10,000 cps and still morepreferably at least about 70,000 cps or more, for example up to about200,000 cps or about 250,000 cps, or about 300,000 cps or more, at ashear rate of 0.1/second. The present drug delivery systems not onlyhave the relatively high viscosity as noted above but also have theability or are structured or made up so as to be effectively placeable,e.g., injectable, into a posterior segment of an eye of a human oranimal, preferably through a 27 gauge needle, or even through a 30 gaugeneedle. Low molecular weight and cross-linked hyaluronic acids exhibitlittle if any shear thinning and can be useful to prepare formulationswithin the scope of our invention, as low viscosity formulations.

The presently useful viscous carrier preferably is a shear thinningcomponent in that as the present composition containing such a shearthinning viscous carrier is passed or injected into the posteriorsegment of an eye, for example, through a narrow space, such as 27 gaugeneedle, under high shear conditions the viscosity of the viscous carrieris substantially reduced during such passage. After such passage, theviscous carrier regains substantially its pre-injection viscosity so asto maintain the microspheres in suspension in the eye.

Any suitable viscous carrier can be used in accordance with the presentinvention. The viscous carrier is present in an amount effective inproviding the desired viscosity to the improved dermal filler.Advantageously, the viscous carrier is present in an amount in a rangeof from about 0.5 wt % to about 95 wt % of the drug delivery system. Thespecific amount of the viscous carrier used depends upon a number offactors including, for example and without limitation, the specificviscous carrier used, the molecular weight of the viscous carrier used,the viscosity desired for the present drug delivery system beingproduced and/or used and like factors.

Examples of useful viscous carriers include, but are not limited to,hyaluronic acid, carbomers, polyacrylic acid, cellulosic derivatives,polycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharides,polyacrylamide, polyvinyl alcohol, polyvinyl acetate, derivativesthereof and mixtures thereof.

Preferably, a dermal filler is used as the viscous carrier. Suitabledermal fillers for that purpose include collagen (sterile collagen issold under the trade names ZYDERM®, ZYPLAST®, COSMODERM®, COSMOPLAST®and Autologen), HYLAFORM® (hyaluronic acid), RESTYLANE® (hyaluronicacid), SCULPTRA® (polylactic acid), RADIESSE® (calcium hydoxyl apatite)and JUVEDERM® . JUVEDERM® , available from Allergan, Inc. (Irvine,Calif.) comprises a sterile, biodegradable, non-pyrogenic, viscoelastic,clear, colorless, homogenized gel consisting of cross-linked hyaluronicacid formulated at a concentration of 24 mg/ml in a physiologic buffer.Hyaluronic acid is a polysaccharide found in the dermis of all mammals.

The molecular weight of the presently useful viscous carrier can be in arange of about 10,000 Daltons or less to about 2 million Daltons ormore. In one particularly useful embodiment, the molecular weight of theviscous carrier is in a range of about 100,000 Daltons or about 200,000Daltons to about 1 million Daltons or about 1.5 million Daltons. Again,the molecular weight of the viscous carrier useful in accordance withthe present invention may vary over a substantial range based on thetype of viscous carrier employed, and the desired final viscosity of thepresent drug delivery system in question, as well as possibly one ormore other factors.

In one very useful embodiment, the viscous carrier is a polymerichyaluronate component, for example, a metal hyaluronate component,preferably selected from alkali metal hyaluronates, alkaline earth metalhyaluronates and mixtures thereof, and still more preferably selectedfrom sodium hyaluronates, and mixtures thereof. The molecular weight ofsuch hyaluronate component preferably is in a range of about 50,000Daltons or about 100,000 Daltons to about 1.3 million Daltons or about 2million Daltons. In one embodiment, the present compositions include apolymeric hyaluronate component in an amount in a range about 0.05% toabout 0.5% (w/v). In a further useful embodiment, the hyaluronatecomponent is present in an amount in a range of about 1% to about 4%(w/v) of the composition. In this latter case, the very high polymerviscosity forms a gel that slows particle sedimentation rate to theextent that often no resuspension processing is necessary over theestimated shelf life, for example, at least about 2 years, of the drugdelivery system. Such a drug delivery system can be marketed inpre-filled syringes since the gel cannot be easily removed by a needleand syringe from a bulk container.

The improved dermal filler of our invention can be administered by anysuitable method as determined by the attending physician. The methods ofadministration permit the improved dermal filler to be administeredlocally to a selected target tissue. Methods of administration includeinjection of a solution or composition containing the dermal filler.

The amount of the neurotoxin contained by the improved dermal filler canbe varied based upon criteria such as the location of the treatment, orthe solubility characteristics of the agent or formulation chosen, aswell as the age, sex, weight, health of the patient and the depth andextent of the facial wrinkles to be treated. Methods for determining theappropriate route of administration and dosage are generally determinedon a case by case basis by the attending physician. Such determinationsare routine to one of ordinary skill in the art (see for example,Harrison's Principles of Internal Medicine (1998), edited by AnthonyFauci et al., 14^(th) edition, published by McGraw Hill).

Our improved dermal filler as well as a botulinum neurotoxin can alsocomprise of one or more of the following non-limiting, exemplary agent(and/or formulation thereof): corticosteroid; botanical agent; wrinklemodifier; growth factor; moisturizer; peptide; antioxidant; keratolytic;retinoid; deoxyribonucleic acid; acne agent; enzyme; ascorbic acid;anticellulite; glycosaminoglycan; facial emollient, and; an anestheticsuch as lidocaine

The amount of the botulinum neurotoxin administered with the viscouscarrier according to a method within the scope of our invention can varyaccording to the particular facial area being treated. Typically, noless about 5 units of a botulinum toxin type A (such as BOTOX® orXeomin®) is administered per injection site (i.e. to each muscle portioninjected), per patent treatment session. For a botulinum toxin type Asuch as DYSPORT®, preferably no less than about 20 units the botulinumtoxin type A are administered per injection site and for a botulinumtoxin type B such as MYOBLOC®, preferably no less than about 200 unitsof the botulinum toxin type B are administered per injection site.Generally, the total amount of BOTOX®, Xeomin®, DYSPORT® or MYOBLOC®,suitable for administration to a patient with an improved dermal filleraccording to the methods of the invention disclosed herein should notexceed about 100 units, about 400 units or about 4000-5000 unitsrespectively, per treatment session.

EXAMPLES

The following examples illustrate aspects of our invention.

Example 1 Low Viscosity Botulinum Toxin-Hyaluronic Acid Formulation

An improved botulinum toxin-hyaluronic acid dermal filler formulationcan be prepared as follows. 1 gram of 1,4-butanediol diglycidyl ether(as cross linker) is added to a 1-L aqueous solution containing 10 ghyaluronic acid (as the viscous carrier), adjusted to pH 12 whilevortexing. The molecular weight of the uncross linked hyaluronic acid isabout 500,000 Daltons. The reaction mixture is incubated at 60° C. for45 minutes and neutralized with glacial acetic acid. The resultingcrosslinked hyaluronic acid can have a crosslinking density of about10%. Ten milligrams of the crosslinked hyaluronic acid is added to 1 mLof an aqueous solution containing 9 mg sodium chloride, 5 mg humanalbumin USP and 1,000 mouse LD₅₀ units of botulinum toxin type Acomplex. The final solution is lyophilized in a 6-mL type I glass vial.An aliquot of the lyophilized formulation containing 100 mouse LD50units of toxin and 1 mg of the crosslinked hyaluronic acid isreconstituted with 1 mL of water for injection (WFI) or with saline forinjection. The resulting solution has a hyaluronic acid concentration ofabout 0.1 wt % and a viscosity of about 300 cps.

Example 2 Low Viscosity Botulinum Toxin-Hyaluronic Acid Formulation witha Higher Hyaluronic Acid Concentration

Another improved botulinum toxin-hyaluronic acid dermal fillerformulation can be prepared as follows. 1 gram of divinyl sulfone (ascross linker) is added to a 500 mL aqueous solution containing 10 ghyaluronic acid (as the viscous carrier) adjusted to pH 14 whilevortexing. The molecular weight of the uncross linked hyaluronic acid isabout 200,000. The reaction mixture is incubated at 40° C. for 8 hoursand neutralized with glacial acetic acid. The resulting crosslinkedhyaluronic acid can have a crosslinking density of about 7%. Twentymilligrams of the crosslinked hyaluronic acid is added to 1 mL of anaqueous solution containing 9 mg sodium chloride, 5 mg human albumin USPand 1,000 mouse LD₅₀ units of botulinum toxin type A complex. The finalsolution is lyophilized in a 6-mL type I glass vial. An aliquot of thelyophilized formulation containing 100 mouse LD50 units of toxin and 1mg the crosslinked hyaluronic acid is reconstituted with 1 mL of waterfor injection (WFI) or with saline for injection. The resulting solutionhas a hyaluronic acid concentration of about 0.5 wt % and a viscosity ofabout 300 cps. Since the amount of cross linking is decreased in theExample 2 formulation the concentration of the hyaluronic acid in theformulation is increased to provide the same viscosity as the Example 1formulation

Example 3 High Viscosity Botulinum Toxin-Hyaluronic Acid Formulation

A high viscosity botulinum toxin-hyaluronic acid dermal fillerformulation can have the ingredients shown in Table 1 below.

TABLE 1 Ingredient Amount Botulinum toxin type A 1000 units Sodiumhyaluronate (polymeric) 2.5% (w/v) Sodium chloride 0.63% (w/v) dibasicsodium phosphate, 0.30% (w/v) heptahydrate Monobasic sodium phosphate,0.04% (w/v) monohydrate Water for Injection q.s. Viscosity at shear rate0.1/second 170,000 ± 25% cps at 25° C.

Preferably the botulinum toxin used is BOTOX®, which is a lyophilized,powdered form of a botulinum toxin type A stabilized with albumin andsodium chloride. The formulation is made by first reconstituting thepowdered botulinum toxin with isotonic saline (sodium chloride) (therebymaking part 1). The sodium hyaluronate can be purchased as a sterilepowder or sterilized by filtering a dilute solution followed bylyophylization of the sodium hyaluronate to yield a sterile sodiumhyaluronate powder. The sterile sodium hyaluronate is dissolved in waterto make an aqueous concentrate (thereby making part 2). Part 1 and part2 are then mixed together. Thus, the reconstituted botulinum toxin ismixed with the sodium hyaluronate concentrate to form a gel, and thephosphate buffers are then added. Water is added q.s. (quantum sufficit,as much as suffices, in this case as much as is required to prepare thehomogenous mixture, dispersion, gel or suspension) and the mixture ismixed until homogenous. The formulation so prepared can be locallyinjected using a 27 gauge or a 30 gauge needle to provide the desiredcosmetic effect.

The sodium hyaluronate powders used in the formulation has a watercontents in a range of about 4% to about 20%, preferably about 4% toabout 8%, by weight. Because the formulation has a density of about 1gm/ml, the percentages set forth herein as being based on weight pervolume (w/v) can also be considered as being based on weight per weight(w/w).

The formulation of Examples 3 uses a sufficient concentration of highmolecular weight, sodium hyaluronate so as to form a gelatinous plug ordrug depot upon local, subdermal injection of the formulation.Preferably the average molecular weight of the hyaluronate used is lessthan about 2 million, and more preferably the average molecular weightof the hyaluronate used is between about 1.3 million and 1.6 million.Since sodium hyaluronate solutions are subject to dramatic shearthinning, these formulations are easily injected through 27 gauge oreven 30 gauge needles. Low molecular weight and cross-linked hyaluronicacids exhibit little if any shear thinning and can be useful to prepareformulations within the scope of our invention, as low viscosityformulations.

The most preferred viscosity range for the formulation is 140,000 cps to280,000 cps at a shear rate 0.1/second at 25° C.

The formulation is formulated using excipients that are fullybiocompatible (i.e. non-toxic) and is buffered at physiological pH bythe low concentration of sodium phosphate salts; rendered isotonic withsodium chloride, and use Water for Injection, USP.

Example 4 Botulinum Toxin Type A-Hyaluronic Acid Dermal FillerFormulation

An improved dermal filled can be made by incorporating a neurotoxin intoa crosslinked sodium hyaluronate matrix as follows. A 10-mL normalsaline solution (0.9% sodium chloride) containing 50 mg albumin humanUSP is first prepared at room temperature. A small amount of botulinumtype A neurotoxin complex, approximately 0.09 micrograms in an aqueoussuspension, is then added to the saline/albumin solution (part I).Separately a 20-mL hydrogel is made with 3% crosslinked hyaluronic acidwith known molecular weight and crosslinking density at pH 7.2 (partII). The part I solution is poured into the part II hydrogel, and theresulting mixture (30 mL in total weight) is gently stirred. A 1-galiquot of the final neurotoxin-containing hydrogel is filled into asyringe and stored in a refrigerator. Alternatively, the 30-mLneurotoxin-containing hydrogel can be filled in glass vials, each with a1-g aliquot, and lyophilized for long-term storage in a freezer.

Example 5 Tetrodotoxin-Hyaluronic Acid Dermal Filler Formulation

Another improved dermal filler can be made by incorporating a neurotoxininto a crosslinked sodium hyaluronate matrix as follows. Ten microgramsof tetrodotoxin is added into a 10-mL hydrogel containing 2% hyaluronicacid with desired molecular weight and crosslinking density or into a10-mL sample of a commercially available, hyaluronic acid-based dermalfiller (i.e. JUVEDERM®) that is either monophasic or particulate-based.The resulting tetrodotoxin-containing hydrogel is then stored in a glassvial with or without lyophilization.

Example 6 High Viscosity Botulinum Toxin-Hyaluronic Acid Formulation

A further improved dermal filled can be made by incorporating aneurotoxin into a crosslinked sodium hyaluronate matrix as follows. ABOTOX® vial containing 100 units of botulinum neurotoxin type A complexis reconstituted with 1 mL normal saline, which is added to 10 mL of ahydrogel containing 2.2% crosslinked hyaluronic acid with desiredviscoelastic properties. The mixture is gently stirred for 10 minutes toachieve homogeneity, and is stored in a refrigerator.

Example 7 In Vivo Diffusion of Biologic-Hyaluronic Acid Formulations

An animal (rat) model was developed to assess the migration ofhyaluronic aid in tissues. A cross-linked hyaluronic acid (JUVEDERM®)was complexed with the contrast agent albumin-gadolinium (MW 65,000daltons, obtained form Biopal, Worcester, Mass.). This contract agent isvisible with magnetic resonance imaging (MRI) in live animals. Previousstudies have shown that the contrast agent does not diffuse out of thehyaluronic acid polymer for a minimum of 48 hours. Therefore, when asignal is visible by MRI following injection, the signal shows thelocation of the hyaluronic acid in the tissue.

A rat was placed under general anesthesia with an inhalational agent. Afive microliter injection of the hyaluronic acid/contrast agent complexwas performed with a 30 G needle in the MGL (tripennate gastrocnemiuslateralis) muscle group of the gastrocnemius muscle. The needle wasplaced 2 cm from the heel, lateral to the tibia with an injection depthof 4-5 mm. There was no movement of the complex during the 6 houranesthesia (FIG. 1). Since there was no contraction of the gastrocnemiusmuscle during anesthesia, there were no mechanical stresses to theinjected area to cause migration of the hyaluronic acid/contrast agentcomplex. The animal was brought out of anesthesia and allowed to performnormal activities with ambulation. The animal was rescanned by MRI after4 hours and 28 hours to determine if there was any migration of thehyaluronic acid complex with the mechanical stress of musclecontraction. The MRI scans showed movement of the hyaluronicacid/contrast agent complex once the animal was awakened and allowed toambulate (FIG. 2). The hyaluronic acid migrated along the musclestriations and eventually moved out of the muscle to the calcanealtendon (Achilles tendon).

The conclusions from the imaging studies were that hyaluronic acid inmuscle remains in the area of injection when the muscle is paralyzed, inthis case, through immobilization with general anesthesia. When musclecontraction was allowed, these mechanical stresses led to migration ofthe injected hyaluronic acid away from the injection site. Theimplication is that local paralysis of the muscle through theco-administration of a botulinum toxin with a dermal filler can increasethe duration of action of the hyaluronic acid in areas of highmechanical stress, such as the perioral area.

Example 8 Facial Mould Method for Determining Extended Duration Effectof Improved Dermal Filler

A female patient 46 years of age presents with bilateral, symmetricaland moderately severe forehead lines during maximum voluntarycontraction of the frontalis muscle. The patient also has, with the lipstogether and at rest, prominent downturned mouth corners.

All make-up and cosmetics are removed from the patient's forehead, whichis then cleansed with an alcohol solution. A silicon replica is made ofthe patients right frontalis during maximum voluntary contraction of thefrontalis muscle as follows. The frontalis muscle is identified byhaving the patient look up and elevate her eyebrows. sEMG is used toconfirm frontalis contraction. An adhesive ring 2.4 cm in diameter ispositioned over an injection site on the right frontalis. A thin layerof freshly prepared silicon replica mixture (rubber silicon, 2 g, andamyl acetate catalyst, 2 drops) is applied within the adhesive ring onthe right side of the forehead during maximum voluntary contraction ofthe frontalis muscle. The patient is instructed to maintain maximalfrontalis muscle contraction for four minutes in which time the siliconepolymer sets. After about 5 minutes, the hardened silicon replica isremoved. The skin surface replica obtained provides a baseline negativeimpression (a mould) and record of the skin surface to which thesilicone polymer set.

A 30 gauge syringe containing 1.5 ml of the cross linked hyaluronic aciddermal JUVEDERM® filler is directed across the left side frontalismuscle fibers perpendicular to the forehead skin surface and keeping theneedle-tip bevel side up, and with the frontalis at rest the JUVEDERM®is injected on the left side of the forehead into the frontalis muscle,at a position 2.5 cm above the superior arch of the left eyebrow, inline with the vertical axis of the center of the pupils. At the sametreatment session a separate 30 gauge syringe containing 1.5 ml of thecross linked hyaluronic acid dermal JUVEDERM® filler mixed with 20 U ofa botulinum toxin type A (BOTOX®) is directed across the right sidefrontalis muscle fibers perpendicular to the forehead skin surface andkeeping the needle-tip bevel side up, and with the frontalis at rest theJUVEDERM® -BOTOX® mixture is injected on the right side of the patient'sforehead into the frontalis muscle, at a position 2.5 cm above thesuperior arch of the right eyebrow, in line with the vertical axis ofthe center of the pupils. The patient is followed over a 104 week periodsubsequent to the injections and at each visit additional rightfrontalis silicon replicas are made.

The baseline silicon replica is compared to the subsequent series ofreplica obtained from the right and left sides of the patient'sforehead. Thus, a silicon replica is placed on a horizontal surface on atable under a digital imaging camera held up by support. The replica isilluminated by light from a light source orientated at an angle (35° isa preferred angle) from the horizontal (and perpendicular to the majorskin lines) thereby generating shadows due to the negative impressionsof lines, wrinkles and furrows in the skin present on the replicasurface. The digital camera is connected by means to a computer equippedwith, for example, Quantirides software (version 2.0, Monaderm, Monaco).The Quantirides software can generate and analyze the imaged skinsurface topography impression, as shown by the silicon replica. Thefollowing parameters can be calculated by the software: mean depth (μm),mean length (mm), total length (mm), number of wrinkles, surface area ofwrinkles (depth.times.length; mm²) and form factor (ratio length/depth)and used to obtain comparative data. The data can show that on theJuvedermTM side of the forehead the wrinkles return to their baselinetopography after 9 to 12 months, but that on the JUVEDERM® -BOTOX® sideof the forehead the wrinkles return to their baseline topography after15 to 18 months.

The same comparative study can be carried out on a patients by left sidevs right side perioral injection of either 1 ml JUVEDERM® or 1 mlJUVEDERM® mixed with 15 units of -BOTOX® and demonstrate on theJUVEDERM® perioral side the downtumed mouth lines (wrinkles) return totheir baseline topography after 6 to 9 months, but that on the JUVEDERM®-BOTOX® side of the mouth the wrinkles do not return to their baselinetopography until 12-15 months post-injection.

Example 9 sEMG Method for Determining Extended Duration Effect of anImproved Dermal Filler Frontalis Muscle

A male patient 54 years of age presents with bilateral, symmetrical andmoderately severe forehead lines during maximum voluntary contraction ofthe frontalis muscle. The patient also has, with the lips together andat rest, prominent downturned mouth corners.

All make-up and cosmetics are removed from the patient's forehead, whichis then cleansed with an alcohol solution. Two pairs of surface EMGelectrodes placed on the left and right frontalis and the monitor of thesEMG processor is placed within the patient's field of vision to enablethe amplitude of the signal to be viewed by the patient and therebyassist with maintenance of maximum voluntary contraction.

The first electrode is placed 2 cm above the brow in a vertical linewith the pupil. The second electrode is positioned laterally to thefirst electrode at a 45-degree angle. The inter-mid-electrode distanceis 1 cm. The second electrode is placed at a 45-degree angle to beparallel with the frontalis muscle fibers to increase recordingaccuracy. The 45-degree angle is measured using a protractor. Therecording electrodes is trimmed for ease of inter electrode spacing. Theground electrodes are placed directly in front of each ear, in thepre-auricular area.

Surface electromyographic quantification of the frontalis muscleactivation is recorded using a Neuroeducator III Surface EMG Processor.The EMG processor has independent isolated channels, each withdifferential amplifiers to enhance the signal to noise ratio andminimize electrical noise and 50 Hertz (Hz) artifact interference.Muscle (electrical) activity is recorded using a continuous analogintegrator, read by the processor at 100 times per second, with apassband of 10-1000 Hz, assuring wideband monitoring without loss of themuscle signal. The recorded sEMG signal is full-wave rectified, and theintegrated sEMG recording is displayed on the screen and stored in bothgraphic and numerical forms.

The same sEMG processor and disposable self-adhesive, pre-gelled Ag—AgClsurface electrodes (1 cm in diameter recording area) are used for allmeasurements. The active and reference electrodes are identicaldisposable adhesive electrodes used to record the amplitude muscleactivity during maximum voluntary contraction. A new set of electrodescan be used for each patient at each visit. Additional sets are used asrequired to maintain good adhesion to the skin of the patient and tominimize 50 hertz Hz noise.

The method of recording enables common mode rejection by the sEMGprocessor, a technique that minimizes crosstalk influences on the muscleactivity recorded. Prior to application of the electrodes, the skin iscleansed with alcohol to minimize 50 Hz skin impedance.

sEMG is carried out during maximum voluntary contraction of thefrontalis muscle using a bipolar surface recording method and the roomtemperature can be maintained at approximately 20° C.

The patient is sitting in an upright relaxed position facing the sEMGmonitor. This positioning can allow the patient to observe their maximumamplitude signal displayed on the monitor and assist in maintainingmaximum voluntary contraction for the required duration. The patient isasked raise his eyebrows to achieve the maximum target signal andsustain it at that level for 10 seconds. The sEMG signal obtained fromthe surface electrodes is processed by computer. The intensity of theresponses is collected during maximum voluntary contraction of thefrontalis muscle.

Surface Electromyography (sEMG) is carried out by comparing baselinesEMG studies with the results of serial sEMG studies following injectionof a dermal filler with or without a botulinum toxin (see below) intothe frontalis muscle. The amplitude (μV) of the maximum voluntarycontraction for the frontalis muscle is obtained by the sEMG recording.The Neuroeducator III surface EMG processor provides an integrated sEMGamplitude value (in μV) recorded from the electrodes placed on the rightand left frontalis muscle. The sEMG recording decreases as the toxinbegins its paralytic effect and increases as the effect of the toxinwears off.

A 30 gauge syringe containing 1.5 ml of the cross linked hyaluronic aciddermal JUVEDERM® filler is directed across the left side frontalismuscle fibers perpendicular to the forehead skin surface and keeping theneedle-tip bevel side up, and with the frontalis at rest the JUVEDERM®is injected on the left side of the forehead into the frontalis muscle,at a position 2.5 cm above the superior arch of the left eyebrow, inline with the vertical axis of the center of the pupils. At the sametreatment session a separate 30 gauge syringe containing 1.5 ml of thecross linked hyaluronic acid dermal JUVEDERM® filler mixed with 20 U ofa botulinum toxin type A (BOTOX®) is directed across the right sidefrontalis muscle fibers perpendicular to the forehead skin surface andkeeping the needle-tip bevel side up, and with the frontalis at rest theJUVEDERM® -BOTOX® mixture is injected on the right side of the patient'sforehead into the frontalis muscle, at a position 2.5 cm above thesuperior arch of the right eyebrow, in line with the vertical axis ofthe center of the pupils. The patient is followed over a 104 week periodsubsequent to the injections and at each visit additional rightfrontalis silicon replicas are made.

The baseline sEMG is compared to the subsequent series of sEMGrecordings obtained from the right and left sides of the patient'sforehead. The recordings can show that on the Juvederm™ side of theforehead the wrinkles return to their baseline topography after 10 to 14months, but that on the Juvederm™-BOTOX® side of the forehead thewrinkles return to their baseline topography after 16 to 19 months

The same comparative study can be carried out on the patient by leftside vs right side perioral injection of either 1 ml JUVEDERM® or 1 mlJUVEDERM® mixed with 15 units of -BOTOX® and demonstrate on theJUVEDERM® perioral side the downturned mouth lines (wrinkles) return totheir baseline sEMG after 8 to 11 months, but that on the JUVEDERM®BOTOX® side of the mouth the wrinkles do not return to their baselinetopography until 14-17 months post-injection.

Example 10 Photographic Method for Determining Extended Duration Effectof an Improved Dermal Filler

A female patient 61 years of age presents with bilateral, symmetricaland moderately severe forehead lines during maximum voluntarycontraction of the frontalis muscle. The patient also has, with the lipstogether and at rest, prominent downturned mouth corners.

All make-up and cosmetics are removed from the patient's forehead, whichis then cleansed with an alcohol solution.

A 30 gauge syringe containing 1.5 ml of the cross linked hyaluronic aciddermal JUVEDERM® filler is directed across the left side frontalismuscle fibers perpendicular to the forehead skin surface and keeping theneedle-tip bevel side up, and with the frontalis at rest the JUVEDERM®is injected on the left side of the forehead into the frontalis muscle,at a position 2.5 cm above the superior arch of the left eyebrow, inline with the vertical axis of the center of the pupils. At the sametreatment session a separate 30 gauge syringe containing 1.5 ml of thecross linked hyaluronic acid dermal JUVEDERM®- filler mixed with 20 U ofa botulinum toxin type A (BOTOX®) is directed across the right sidefrontalis muscle fibers perpendicular to the forehead skin surface andkeeping the needle-tip bevel side up, and with the frontalis at rest theJUVEDERM®-BOTOX® mixture is injected on the right side of the patient'sforehead into the frontalis muscle, at a position 2.5 cm above thesuperior arch of the right eyebrow, in line with the vertical axis ofthe center of the pupils. The patient is followed over a 104 week periodsubsequent to the injections and at each visit additional photographsare taken as set forth below.

Photographs are taken of the patient before injection (baseline) and atand an regular one week intervals; for 104 weeks after injection. Ateach visit, digital and 35 mm photographs frontal view of the patient'supper face are taken.

The patient is positioned in the same manner for all photographs. Astereotactic device is used to ensure consistent positioning of the facein relation to the camera which comprises a dedicated chin/head supportassembly. In addition, the image obtained at the screening visit (dayzero) is used as a reference to ensure identical positioning of the headat all subsequent visits. Following positioning of the patient andverification of the set-up of the camera, the patient is requested tomaximally elevate her eyebrows (by maximum voluntary contraction of thefrontalis muscle) by viewing the fixed indicator. Three exposures of thefull frontal view (0°) of the upper face can then be taken with both a35 mm and with a digital camera.

For all photographs lighting, framing and exposure ratios are heldconstant. Standardized magnification and aperture can also be used. Formagnification a standardized reproduction ratio of 1:5 (35 mmequivalent) is used for both the digital and 35 mm facial photographs.The camera aperture for all 35 mm facial photographs is at f/16, and forall digital facial photographs the camera aperture is set at f/32.

The 35 mm photographic images are digitally scanned and analyzed in thesame way as the digital photographs. All photographic images arecalibrated and analyzed using both Mirror DPS (Canfield Scientific,Inc., Fairfield, N.J.) and Image Pro Plus (Media Cybernetics, SilverSpring, Md.). The software can draw a horizontal line through the innercanthus of the eyes and calculate the distance in millimeters betweenthis line and the lower edge of the eyebrow at three specific points.Images from a patient are re-sized and adjusted to the samemagnification as the baseline image using Mirror DPS, i.e. all imagesfor a patient is identically sized. Images are then exported to ImagePro Plus and rotated such that a straight blue line intersects the innercanthus of the eyes.

A reduction in brow mobility (in mm) during maximal voluntarycontraction is used to show onset, peak and duration of the paralyticeffect. Photography is carried out by comparing baseline 2 dimensionaldigital (2D) and 35 mm image studies with results of serial 2D and 35 mmimage studies following injection of into the frontalis muscle.

Response is determined by comparing baseline 2 dimensional digital (2D)and 35 mm image studies with results of serial 2D and 35 mmphotographical image studies following injection of a dermal filler withor without a botulinum neurotoxin into the frontalis muscle (see below).The reduction of the upward mobility of the eyebrow measured duringmaximum eyebrow elevation is obtained using the following measurement.The parameters determined by the data from this photography analysis areonset of wrinkle attenuation, extent of wrinkle alleviation and durationof the wrinkle attenuation or alleviation.

The baseline photographs are compared to the subsequent series ofphotographs taken of the right and left sides of the patient's forehead.The images can show that on the Juvederm™ side of the forehead thewrinkles return to their baseline topography after 9 to 12 months, butthat on the Juvederm™-BOTOX® side of the forehead the wrinkles return totheir baseline topography after 15 to 18 months.

The same comparative study can be carried out on the patient by leftside vs right side perioral injection of either 1 ml Juvederm™ or 1 mlJuvederm™mixed with 15 units of -BOTOX® and demonstrate on the Juvederm™perioral side the downturned mouth lines (wrinkles) return to theirbaseline sEMG after 8 to 11 months, but that on the Juvederm™-BOTOX®side of the mouth the wrinkles do not return to their baselinetopography until 14-17 months post-injection.

All references, articles, patents, applications and publications setforth above are incorporated herein by reference in their entireties.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

We claim:
 1. A method for treating a facial wrinkle by administering tothe patient a pharmaceutical composition comprising a) a botulinumneurotoxin, b) a serum albumin, wherein the concentration of the serumalbumin in the pharmaceutical composition is about 5 μg or less per unitof the botulinum neurotoxin, and c) a viscous carrier comprising across-linked hyaluronic acid, wherein said cross-linked hyaluronic acidhas a cross-linking density of from 5% to about 10%, wherein thepatient's facial wrinkles are alleviated for a longer period of timethan they are by administration of a pharmaceutical composition whichdoes not comprise a botulinum neurotoxin.
 2. The method of claim 1,wherein the administering is carried out by local subdermal injection ofthe pharmaceutical composition.
 3. The method of claim 1, wherein theperiod of alleviation of the facial wrinkles is determined by a methodchosen for the group of method consisting of facial mould,electromyographic (EMG) recording and photography.
 4. The method ofclaim 1, wherein the period of time of alleviation of the facialwrinkles is from about 20% to about 100% longer than the period of timethe patient's facial wrinkles are alleviated by administration of apharmaceutical composition injected in the same volume into the samepatient at the same location, to treat the same facial wrinkles, andwhich contains the same amount of the botulinum neurotoxin, but whichdoes not comprise a viscous carrier comprising the cross-linkedhyaluronic acid.
 5. The method of claim 3 wherein the hyaluronic acidhas a molecular weight between about 10,000 Daltons and about 20 millionDaltons.
 6. The method of claim 3 wherein the concentration ofcross-linked hyaluronic acid in the pharmaceutical composition isbetween about 0.1 wt % and about 99 wt %.
 7. The method of claim 1wherein the viscosity of the pharmaceutical composition is between about100 cps and about 300,000 cps at 25° C., at a shear rate of 0.1 /second.8. The method of claim 1 wherein the botulinum neurotoxin is a botulinumneurotoxin type A.
 9. A method for treating a facial wrinkle by local,subdermal administering to the patient a pharmaceutical compositioncomprising a) a botulinum neurotoxin type A, b) a serum albumin, whereinthe concentration of the serum albumin in the pharmaceutical compositionis about 5 μg or less per unit of the botulinum neurotoxin, and c)cross-linked hyaluronic acid, wherein said cross-linked hyaluronic acidhas a cross-linking density of from 5% to about 10%, wherein thepatient's facial wrinkles are alleviated for a period of time which isfrom about 20% to about 100% longer than the period of time thepatient's facial wrinkles are alleviated by administration of a secondpharmaceutical composition injected in the same volume into the samepatient at the same location, to treat the same facial wrinkles, andwhich second pharmaceutical composition contains the same amount of thebotulinum neurotoxin type A, but which does not comprise thecross-linked hyaluronic acid.
 10. The method of claim 1 wherein theconcentration of the botulinum neurotoxin in the pharmaceuticalcomposition is about 100 units/mL.
 11. The method of claim 1 wherein theconcentration of the serum albumin in the pharmaceutical composition isabout 5 μg per unit of the botulinum neurotoxin.
 12. The method of claim1 wherein the concentration of the cross-linked hyaluronic acid in thepharmaceutical composition is between 20 mg/mL to 40 mg/mL.
 13. Themethod of claim 1 wherein the amount of the cross-linked hyaluronic acidin the pharmaceutical composition is at least 85%.
 14. The method ofclaim 1 wherein the pharmaceutical composition further comprises anuncross-linked hyaluronic acid.
 15. The method of claim 9 wherein theconcentration of the botulinum neurotoxin type A in the pharmaceuticalcomposition is about 100 units/mL.
 16. The method of claim 9 wherein theconcentration of the serum albumin in the pharmaceutical composition isabout 5 μg per unit of the botulinum neurotoxin.
 17. The method of claim9 wherein the concentration of the cross-linked hyaluronic acid in thepharmaceutical composition is between 20 mg/mL to 40 mg/mL.
 18. Themethod of claim 9 wherein the amount of the cross-linked hyaluronic acidin the pharmaceutical composition is at least 85%.
 19. The method ofclaim 9 wherein the pharmaceutical composition further comprises anuncross-linked hyaluronic acid.
 20. A method for treating a facialwrinkle by administering to the patient a pharmaceutical compositioncomprising a) a botulinum neurotoxin type A, wherein the concentrationof botulinum neurotoxin type A is about 100 units/mL, b) a serumalbumin, wherein the concentration of the serum albumin in thepharmaceutical composition is about 5 μg or less per unit of theboutulinum neurotoxin type A, and c) a hydrogel comprising cross-linkedhyaluronic acid, wherein said cross-linked hyaluronic acid has across-linking density of from 5% to about 10%, and wherein theconcentration of the cross-linked hyaluronic acid in the pharmaceuticalcomposition is between 20 mg/mL to 40 mg/mL, wherein the patient'sfacial wrinkles are alleviated for a longer period of time than they areby administration of a pharmaceutical composition which does notcomprise a botulinum neurotoxin type A.